Surfactant microbubbles and process for preparing and methods of using the same

ABSTRACT

The invention relates to an ultrasound contrast agent (UCA) comprising an outer shell and a gas core. The gas core is filled with oxygen, and the outer shell comprises a first surfactant and a second surfactant. The invention also relates to a method of making an oxygen-filled UCA and delivering oxygen to a local area of a subject&#39;s body. The method comprises injecting a composition comprising an oxygen-filled UCA of the invention into the subject&#39;s body; directing ultrasound radiation to the local area in an intensity sufficient to rupture the UCA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of, and claimspriority to, U.S. patent application Ser. No. 14/835,636, filed Aug. 25,2015, now allowed, which claims priority under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application No. 62/042,184, filed Aug. 26, 2014,the entire contents of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberW81XWH-11-1-0630 awarded by United States Army Medical Research &Material Command. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The highly chaotic process of tumor angiogenesis (whereby canceroustumors recruit and develop new blood vessels) results in hypoxicconditions on the cellular level, because oxygen transport within thetumor is inadequate to compensate for the high metabolic rate. Whereashealthy subcutaneous tissue generally exhibits oxygen partial pressuresfrom 40-60 mmHg, many cancers exhibit partial pressures between 2 and 18mmHg. Hypoxic cells have been shown to be more resistant to death fromradiation exposure than aerobic cells. This phenomenon provides aninnate level of tumor resistance to radiotherapy and chemotherapy,resulting in decreased tumor response and increased rates of recurrence.Systemic approaches for oxygen (O₂) delivery using hyperbaric chambersfor overcoming tumor hypoxia have shown some promise, but this becomestechnically challenging in conjunction with radiation therapy from atemporal standpoint and localized changes in oxygenation levels havebeen modest.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to an ultrasound contrast agent(UCA) comprising an outer shell and a gas core. The gas core is filledwith oxygen, and the outer shell comprises a first surfactant and asecond surfactant. The purity of the oxygen enclosed in the UCA is inthe range of about 22% to about 100%. The first surfactant isd-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) orpolyoxyethylene sorbitan monooleate, while the second surfactant isselected from the group consisting of: sorbitan esters, alkylphenolethoxylate-based surfactants, alcohol ethoxylate-based surfactants,silicone-based surfactants, alkyl poly(ethylene oxide), alkylphenolpoly(ethylene oxide), copolymers of poly(ethylene oxide) andpoly(propylene oxide), alkyl polyglucosides, fatty alcohols, cocamideMEA, cocamide DEA, and polysorbates. The mean diameter of the UCA is inthe range of 1 μm to 10 μm. The half-life of the UCA under 5 MHzultrasound is in the range of 1 to 20 minutes.

In another aspect, the invention relates to a method of making anoxygen-filled UCA. The method comprises the steps of: mixing a firstsurfactant and a second surfactant in phosphate buffered saline; heatingthe mixture until both surfactants are dissolved; cooling said mixtureto room temperature while stirring; optionally autoclaving the mixtureand cooling the mixture while stirring; placing the vessel containingthe mixture in an ice bath, purging said mixture using a gas whilesonicating said mixture; separating microbubbles; adding a lyoprotectantto the microbubbles; freeze-drying the microbubbles; sealing themicrobubbles under vacuum; and refilling the microbubbles withsubstantially pure oxygen.

In yet another aspect, the invention relates to a method of deliveringoxygen to a local area of a subject's body. The method comprisesinjecting a composition comprising a UCA of the invention into thesubject's body; and directing ultrasound radiation to the local area inan intensity sufficient to rupture the UCA.

In yet another aspect, the invention relates to a method of improvingeffectiveness of radiotherapy and chemotherapy against cancer. Themethod comprises the steps of: injecting a composition comprising a UCAof the invention into a subject suffering from cancer; directingultrasound radiation to a location of said cancer in an intensitysufficient to rupture the UCA; and applying radiotherapy and/orchemotherapy.

In yet another aspect, the invention relates to a method of delivering adrug to a subject. The method comprises the steps of: preparing adrug-containing UCA; injecting the UCA into the circulatory system ofthe subject; and directing ultrasound radiation to a location ofinterest with an intensity sufficient to rupture the drug-containingUCA.

In yet another aspect, the invention relates to a method of improve theeffectiveness of a therapeutic agent. The method comprises the steps of:preparing an oxygen-filled UCA; co-injecting the UCA with thetherapeutic agent into the circulatory system of the subject; anddirecting ultrasound radiation to a location of interest with anintensity sufficient to rupture the oxygen-filled UCA.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are certainembodiments of the invention depicted in the drawings. However, theinvention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings.

FIG. 1A is a light microscopy image of SE61_(O2) at 40× magnification(size bar=20 μm).

FIG. 1B depicts size distribution measurement of SE61_(O2) by dynamiclight scattering, showing a mean particle diameter of approximately 3μm.

FIG. 1C depicts flow cytometry results for distinct, separate bubbles(black). Forward scattered light (FSC-A) is represented on the x-axisand particle counts on the y-axis. Populations were observed with atotal bubble population of approximately 6.5×10⁷ microbubbles/ml.

FIG. 1D depicts flow cytometry results for counting bead (red). Forwardscattered light (FSC-A) is represented on the x-axis and particle countson the y-axis. Populations were observed with a total bubble populationof approximately 6.5×10⁷ microbubbles/ml.

FIG. 2A depicts in vitro ultrasound enhancement of SE61_(O2) and thecontrolled microbubbles (control).

FIG. 2B depicts that oxygen-filled microbubbles also showed good overallstability with a half-life of over 15 minutes (half-life thresholddisplayed by dashed line).

FIG. 3 depicts in vitro ultrasound enhancement of SE61_(O2) in a flowphantom using a commercial ultrasound scanner at baseline (top) and 30seconds post injection (bottom).

FIG. 4 depicts relative change in oxygen partial pressure (pO₂) levelsin 100 ml of degassed saline over 20 minutes under different conditions:SE61_(O2) and ultrasound, ultrasound alone, SE61_(O2) alone, andnitrogen filled SE61 with ultrasound (* p<0.0001).

FIG. 5 depicts a schematic diagram of an ultrasound contrast agent (UCA)composition. The core of a UCA can be any gas, air, a drug, or a mixtureof drugs.

FIG. 6 depicts in vivo proof of concept delivery experiments. The topultrasound image shows contrast pO₂ probe arrival within the peripheryof the tumor with slight peripheral enhancement denoted with two arrows.The middle ultrasound image shows flash-destructive ultrasound pulseused to destroy the microbubbles. The bottom ultrasound image shows aslight decrease in peripheral enhancement immediately after microbubbledestruction.

FIG. 7 depicts the intra-tumoral pO2 levels over time for Mouse 1 andMouse 2 after injection of SE61_(O2) with ultrasound, SE61_(O2) alone,and SE61_(N2) with ultrasound.

FIGS. 8A-8E depict in vivo images showing enhancement of the renalvasculature in a 3 kg swine after injection of SE61 filled with O₂.Imaging was performed using a SIEMENS® S3000 ultrasound system with 9MHz linear probe in CPS mode. Microbubble signal is displayed in theleft half of each images, while the normal B-mode ultrasound isdisplayed in the right half of each image. FIGS. 8A, 8B, 8C, 8D, and 8Ewere taken at 19, 26, 34, 44, and 54 seconds after injection,respectively. These data demonstrate the stability of the oxygen filledmicrobubble and ability to deliver oxygen in a large animal model.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

As used herein, the terms “comprises,” “comprising,” “containing,”“having,” and the like can have the meaning ascribed to them in U.S.patent law and can mean “includes,” “including,” and the like.

Unless specifically stated or obvious from context, the term “or,” asused herein, is understood to be inclusive.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (aswell as fractions thereof unless the context clearly dictatesotherwise).

As used herein, “half-life” refers to the amount of time required forhalf of microbubbles ruptured under specific condition.

As used herein, “lyoprotectant” and “cryoprotectant” areinterchangeable. Both refer to a compound that minimizes or preventsstructural and/or functional integrity loss of UCA that can occur duringthe drying stage of a freeze-drying process.

As used herein, the term “substantially pure” refers to a gas having apurity more than 80% weight percentage.

As used herein, the term “hydrophilic-lipophilic balance” (“HLB”) is arelative measure of the ratio of polar and non-polar groups present in asurfactant.

As used herein, the term “microbubble” is used interchangeably with“ultrasound contrast agent”, which is defined below.

As used herein, the term “SPAN®” refers to a sorbitan monoester that isused as a non-ionic detergent, and sold by Sigma-Aldrich.

As used herein, “ultrasound contrast agent” (“UCA”) refers tosurfactant-stabilized gas bubbles.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a gas delivery methodutilizing an ultrasound contrast agent (UCA) to deliver a gas to alocation of a subject's body. The gas can be air, nitrogen, oxygen,nitric oxide, perfluorocarbon, sulfur hexafluoride, carbon dioxide andthe like. In one aspect, the gas delivery method comprises: preparing aUCA with a gas core filled with a specific gas; injecting the UCA into asubject's body; and directing ultrasound radiation to a specificlocation of the subject's body with an intensity sufficient to rupturethe UCA. Thus, the gas inside the UCA will be released to the localtissue of the subject. The invention also includes a UCA suitable foruse in the gas delivery method, a method of making, and a method ofusing the same. Another aspect of the invention includes a method ofsterilization and a method of making a sterilized UCA are included inthe invention.

Composition

In one aspect, the invention includes a UCA comprising an outer shelland a gas core filled with substantially pure gas (FIG. 5). The gasbubble can be stabilized by at least two surfactants.

In one embodiment, the gas is oxygen. Cancer cells living in hypoxicconditions are resistant to radiation exposure. Therefore, deliveringoxygen to cancer cells using the gas delivery method in the inventioncan improve effectiveness of radiotherapy and chemotherapy.

In another embodiment, the gas is nitrogen. In yet another embodiment,the gas is nitric oxide. In yet another embodiment, the gas is a mixtureof nitric oxide and oxygen. Nitric oxide/oxygen blends promote capillaryand pulmonary dilation to treat primary pulmonary hypertension inneonatal patients.

In certain embodiments, the purity of oxygen by volume can be about100%, or at least about 95%, or at least about 90%, or at least about85%, or at least about 80%, or at least 22%.

Surfactants useful in the practice of the invention can include anybiocompatible surfactants known in the art including anionic, cationic,zwitterionic, and non-ionic surfactants. In an embodiment, the UCAcomprises two non-ionic surfactants. When the surfactant is non-ionic,the hydrophilic-lipophilic balance (HLB) of the surfactant is betweenabout 6 and about 16. Non-limiting examples of non-ionic surfactantsuseful in the practice of the invention include, but are not limited to,d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), sorbitanesters, alkylphenol ethoxylate-based surfactants, alcoholethoxylate-based surfactants, silicone-based surfactants, alkylpoly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide), alkyl polyglucosides,fatty alcohols, cocamide MEA, cocamide DEA, and polysorbates.

In certain embodiments, the two non-ionic surfactants are selected fromthe group consisting of sorbitan esters, polysorbates and TPGS. Thepossible compositions in these embodiments include combinations ofsorbitan esters/polysorbates, sorbitan esters/TPGS andpolysorbates/TPGS. The different surfactants in various combinations, invarious concentrations, and encapsulating different gases will dictatethe packing density and strength of inter-molecular forces around thegas.

In another embodiment, the first surfactant is TPGS, and the secondsurfactant is sorbitan esters. In yet another embodiment, the firstsurfactant is polyoxyethylenesorbitan monolaurate, and the secondsurfactant is sorbitan esters.

UCA Size

The UCA in the examples can have a mean diameter in the range of 1 μm toabout 10 μm. In certain embodiments, the UCA in the invention has a meandiameter in range of 2 μm to 7 μm, 2 μm to 6 μm, or 2 μm to 5 μm. In oneinstance, the UCA in the invention has mean diameter of about 3 μm.Although the examples discuss microbubbles only, it is well understoodthat nanobubbles with a mean diameter between 1 nanometer and 1,000nanometers are included in this invention. Mechanistically, nanobubblesand microbubbles function in the same way in terms of delivering gas.

UCA Stability and Functionality

A shelf-stable UCA is preferred for the gas delivery method, as the UCAhas to be stable long enough to pass through the circulatory system andpotentially to the tumor vasculature. In certain embodiments, the UCAcomprises surfactants and a particulate material. In one embodiment, theparticulate material is one of the surfactants. In other embodiments, anexogenous particulate material in incorporated into the UCA. Theexogenous particulate material is incorporated any time prior to thesonication step. Examples of particles that are useful in the practiceof the invention include, but are not limited to: solid surfactant;quantum dots; colloidal gold; carbon nanotubes; carbon nanotubescontaining drug; polystyrene; SPIO (superparamagnetic iron oxide); ironoxide; coated nanoparticles containing a drug; imaging agents such asgas; radiopaque species; MRI contrast agents such as Gadoliniumcompounds; a drug in nanoparticle form (e.g., by grinding);nanocapsules; hollow drug-containing contrast medium containingparticles with attached targeting agents such as antibodies, portions ofantibodies, peptide sequences etc.; viruses; and inactivated viruses.

To test the stability of a UCA, ultrasound radiation under certainintensity is applied to the UCA. In one embodiment, the half-life of theUCA under 0.69 MPa ultrasound is in the range of 1 to 20 minutes. Incertain embodiments, the half-life of the UCA under 0.69 MPa ultrasoundis in the range of 5 to 20 minutes; or in the range of 10 to 20 minutes.In one instance, the half-life of the UCA under 0.69 MPa ultrasound isabout 15 minutes.

Method of Making a UCA

In another aspect, the invention includes a method of making a UCA. Thegeneral steps are the following:

-   -   a. mixing a first surfactant and a second surfactant in        phosphate buffered saline;    -   b. heating the mixture until both surfactants are dissolved;    -   c. cooling said mixture to room temperature while stirring;    -   d. optionally autoclaving the mixture and cooling the mixture        while stirring;    -   e. placing the mixture in a vessel in an ice bath, purging said        mixture using a first gas while sonicating said mixture;    -   f. separating microbubbles;    -   g. adding a lyoprotectant to the microbubbles;    -   h. freeze-drying the microbubbles;    -   i. sealing the microbubbles under vacuum; and    -   j. refilling the microbubbles with a second gas.

In one embodiment, the lyoprotectant is a saccharide added prior tofreeze-drying the UCA. In one embodiment, the saccharide is trehalose;in another embodiment, the saccharide is glucose. In a non-limitingexemplary example, a UCA is diluted in 1:1 by volume with a solution ofthe saccharide. The final concentration of the saccharide in the mixturewith the UCA can be from about 1 millimolar (mM) to less than about 200mM, preferably from about 10 mM to about 140 mM, and more preferablyabout 50 mM to about 100 mM. The mixture is then flash frozen, forinstance, in liquid nitrogen, and then freeze-dried at a temperature ofabout −80° C. to about −70° C. In one embodiment, octafluoropropane gasis the first gas to purge the mixture to form bubbles havingoctafluoropropane gas.

The method described herein can be used to reconstitute a UCA with anygas of choice, including, but not limited to, oxygen, nitrogen, ornitric oxide. An exemplary method of freeze drying and filling UCA withgas is as follows. An aliquot of a UCA suspension is placed in a 15 mllyophilization vial (West Pharmaceutical Services, Lionville, Pa.). AFLUROTEC® lyophilization stopper (West Pharmaceutical Services) isplaced in the neck of the vial up to the first groove. The agent is thenflash frozen in liquid nitrogen. The vial is placed on a previouslychilled (initially to −80° C.) two-shelf stoppering rack of a VIRTIS®Benchtop freeze-dryer (Gardiner, N.Y.) and freeze dried at pressuresbelow 300 μBar and a condenser temperature of −76° C. For gas filling,the piston of the stoppering rack is lowered prior to venting the freezedryer, thus sealing the stopper in the vial under vacuum. The stopperedvial is removed from the freeze dryer, and the gas of choice isintroduced into the individual vial from a tank, passing through asterile filter, via a needle pierced through the stopper septum. A gasflow rate of 50 ml/min for the first 5 to 10 seconds and then 20 ml/minfor the next minute can be used to insure the vial is filled. Forcapsules filled with air, the freeze drier is vented to the atmosphereprior to stoppering the vials.

In one embodiment, oxygen is used to charge a UCA to generate anoxygen-filled UCA. In certain embodiments, the purity of oxygen is about100%, or at least about 95%, or at least about 90%, or at least about85%, or at least about 80%.

In certain embodiments, the gas-filled UCA is reconstituted before useby addition of an aqueous solution. In one embodiment, the aqueoussolution is sterile water. In another embodiment, the aqueous solutionis phosphate buffered saline.

In Vitro Oxygen Release

An oxygen-filled UCA made according to the methods described herein wasconfirmed to rupture and release oxygen under ultrasound radiation.Ultrasound triggering was performed using a SONIXRP® scanner with aPA4-2 cardiac probe operating at 100% acoustic output (approximately 3.6MPa peak-peak pressure) at 4 MHz in power Doppler mode. Two millilitersof UCA was added to 100 ml of degas sed saline. Samples were triggeredwith ultrasound over 20 minutes with readings obtained every 30 seconds.FIG. 4 shows that triggered UCAs release oxygen.

In Vivo Application

In vivo oxygen release immediately prior to therapy has demonstrated anability to elevate hypoxic tumor oxygenation levels in animals (FIGS. 6and 7). This is significant because a relatively small increase inoxygen partial pressure can result in significant sensitization. Therelationship between pO₂ and radiosensitivity has been well studied invitro: while cells at a pO₂ of <2 mmHg are almost completelyradiation-insensitive, cell sensitivity reaches its asymptotic peak atapproximately 30 mmHg. In one embodiment, the method of oxygen releasedescribed herein is used to improve the effectiveness of radiotherapyand chemotherapy against cancer. Such method comprises: preparing anoxygen-filled UCA; injecting the UCA into a cancer patient's body;directing ultrasound radiation to a specific location of the cancerpatient's body with an intensity sufficient to rupture the UCA; andapplying radiotherapy and/or chemotherapy. Thus, the oxygen inside theUCA will be released to the local cancer tissue, and help theeffectiveness of radiotherapy and chemotherapy.

Alternatively, the oxygen-filled UCA can be used to improve theeffectiveness of a therapeutic agent. The method comprises the steps of:preparing an oxygen-filled UCA; co-injecting the UCA with thetherapeutic agent into the circulatory system of the subject; anddirecting ultrasound radiation to a location of interest with anintensity sufficient to rupture the drug-containing UCA.

In another embodiment, the gas delivery method described herein can beused to enhance wound healing and treat wounds and other conditions thatare susceptible through treatment with hyperbaric medicine. Examples ofsuch wounds include ulcers, diabetic ulcers, pressure sores, and thelike.

Following the same mechanism of delivering oxygen to a subject's body,UCA can be used as a drug delivery carrier. The method of deliverycomprises: preparing a drug-contained UCA; injecting the UCA into thecirculatory system of the subject; monitoring the UCA reaching aspecific location of interest; and directing ultrasound radiation tothat location of interest with an intensity sufficient to rupture theUCA. Thus, the drug releases to the location of interest after therupture. A single UCA can contain both a drug and oxygen. Alternatively,a mixture of drug-containing UCAs and oxygen-containing UCAs can beutilized, either in parallel or in series. Drug-containing UCAs andexamples of drugs that can be incorporated in UCAs are described in U.S.Patent Application Publication Nos. 2004/0258760, 2004/0258761,2008/0247957, 2008/0279783, and 2009/0196827.

WORKING EXAMPLES

The invention is further described in detail by reference to thefollowing working examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Thus, the invention should in no way be construed as beinglimited to the following examples, but rather, should be construed toencompass any and all variations which become evident as a result of theteaching provided herein.

Abbreviations Used O₂: Oxygen

pO₂: Partial pressure of oxygenSE61_(O2): Experimental oxygen microbubble consisting of a SPAN®60 andwater soluble vitamin E shell

FE-DBD: Floating Electrode Dielectric Barrier Discharge PTPBS:Plasma-Treated Phosphate-Buffered Saline

SE61: UCA composed of SPAN®60 & water-soluble vitamin EddH2O: Double-distilled WaterNAC: N-acetyl-cysteine

PBS: Phosphate-Buffered Saline DBD: Dielectric Barrier DischargeMaterials Surfactants

Two non-ionic surfactants were used for preparing the microbubbles.SPAN®60 (sorbitan monostearate) (S7010, lot #010M0128) was purchasedfrom Sigma Aldrich (St. Louis, Mo.) and used without furtherpurification. United States National Formulary grade TPGS (batch#78971100) was bought from Eastman Chemical and used without furtherpurification.

Other Chemicals

Octafluropropane (99% min) was purchased from American Gas Group(Toledo, Ohio) and used after passing through a sterile 0.22 μm filter(Nalgene, Rochester, N.Y.) in an aseptic (laminar flow) hood. Pureoxygen gas was purchased from Airgas USA, LLC. (Radnor, Pa.) and usedafter passing through a sterile 0.22 μm filter. Phosphate buffer saline(0.1M) was used as the buffer for preparing and testing the microbubblesafter filtration using a 0.22 μm GV DURAPORE® membrane filter (lot#R6AN42605) purchased from Millipore. Sodium chloride, potassiumchloride, sodium phosphate dibasic and potassium phosphate monobasicwere all obtained from Sigma Aldrich (St. Louis, Mo.). D-glucose(lyoprotectant agent for freeze-drying the microbubbles) and Nile Redwas also obtained from Sigma Aldrich (St. Louis, Mo.). For freeze-dryingand UCA storage, 15 mL lyophilization vials were obtained from WestPharmaceutical Services (Lionville, Pa.).

Methods Microbubble Fabrication

The UCA used in this study (termed SE61_(O2)) was prepared according tothe method described in Solis et al., “Preserving Enhancement inFreeze-Dried Contrast Agent ST68: Examination of Excipients.” Int. J.Pharm. 396:(1-2):30-38 (2010). In summary, SPAN60® (sorbitanmonostearate; 1.5 g) and water soluble vitamin E (d-alpha tocopherylpolyethylene glycol 1000 succinate) (TPGS; 1.3 g) were dissolved in 50ml of phosphate buffered saline (PBS) and autoclaved for 30 minutes. Themixture was cooled under magnetic stirring and placed in an ice bath andcontinuously sonicated for 3 minutes at 110 W using a 0.5-inch probehorn (CL4 tapped horn probe with 0.5″ tip, Misonix Inc., Farmingdale,N.Y.). The solution was purged with a steady stream of octafluoropropanebefore and during the sonication. Microbubbles were extracted from thesolution via gravity separation in a 250 ml glass separation funnel,washed 3 times with cold (4° C.) PBS every 90-120 minutes using the sameseparation funnels. Two milliliter aliquots of native bubble suspensionwere pipetted with a pipet specifically designed for viscous fluids(GILSON® PIPETMAN®, Middleton, Wis.), into 15 ml lyophilization vials.Finally, 0.5 ml 400 mM glucose (Sigma Aldrich) was added to each vial asa lyoprotectant.

The 2.5 ml samples in lyophilization vials were freeze-dried. In brief,FLUORTEC® lyophilization stoppers were inserted into the vials to thefirst groove (leaving a gap for air to escape). The samples wereflash-frozen in liquid nitrogen for 5 minutes and subsequentlyfreeze-dried for 24 hours on a VIRTIS® Benchtop freeze-dryer (Gardiner,N.Y.) fitted with a two shelf assembly that had been previously cooledto −80° C. The conditions during this process were −76.5° C. (in thevacuum drier chamber) and 17-20 μBar. Before removing the samples, thepiston was lowered to depress the stoppers, effectively vacuum sealingthe vials. The gas of choice (nitrogen or oxygen, Airgas LLC, Radnor,Pa.) was introduced through the FLUORTEC® stoppers using a sterilesyringe needle, after passage through a sterile 0.22 μm NALGENE® filter(Nalge Nunc International, Rochester, N.Y.) at an initial flow rate of50 ml/min for 5-10 seconds then 20 ml/minutes for one minute to insurethe vials were filled. The procedure was performed under an asepticlaminar flow hood and vials sealed with parafilm until use. Immediatelyprior to use, the vials were charged with 2 ml of deionized water. Incertain embodiments, the deionized water will be saturated with oxygen.As a control group (to ensure all ultrasound enhancement is attributedto the addition of O₂), samples were maintained under vacuum andreconstituted in the usual way with deionized water prior to use(thereby creating bubbles without additional gas).

Particle Morphology, Size, and Concentration Characterization

Particle morphology was assessed by light microscopy using an OLYMPUS®IX71 microscope (Tokyo, Japan) at 40× magnification. The sizedistribution of the microbubbles was analyzed using a ZETASIZER® Nano ZS(Malvern Instruments, Worcestershire, UK) in Z-average mode, usingdynamic light scattering techniques. Using this technique, a 50 μlsample was dispersed in 950 μl PBS in tapered cuvettes forsize-analysis. Particle counting was performed using a flow cytometer,LSRII (BD Biosciences, San Jose, Calif.). With this technique, 10 μl ofmicrobubbles were added to 0.5 ml of deionized water and 10 μl of UVCOUNTBRIGHT® Absolute Counting Beads (containing 9,800 beads used as acounting standard; Life Technologies, Grand Island, N.Y.). Countingbeads and SE61_(O2) particles were separated using FSC-A and PE-Afilters.

UCA Stability and Ultrasound Enhancement Characterization

A custom-built acrylic plastic sample holder with a clear acousticwindow (2.5 cm×2.5 cm) was placed in a tank filled with 75 liters ofdeionized water (temperature-controlled to 37° C.) for in vitro acoustictesting of the UCA. A single element 5 MHz transducer (Panametrics,Waltham, Mass.; 12.7 mm diameter, −6 dB bandwidth of 91% and focallength of 50.8 mm) was focused through the acoustic window using an x-ypositioning system (Edmund Scientific, Barrington, N.J.) and a 5072pulser-receiver (Panametrics) was used to generate acoustic pressures of0.69 MPa peak negative pressure with a pulse repetition frequency of 100Hz. This ultrasound signal is expected to be strong enough to detectmicrobubble response, but not expected to generate inertial cavitation.A magnetic stir bar constantly recirculated the bubbles through thefocus of the transducer. Reflected signals from the UCA were detected bythe same transducer and amplified 40 dB before being read by a digitaloscilloscope (LECROY® 9350A, LeCroy Corporation, Chestnut Ridge, N.Y.).Data acquisition and processing was done on a computer with LABVIEW® 7.1(National Instruments, Austin, Tex.).

To determine the dose dependence of ultrasound signal enhancement, acumulative dosage response curve was generated for UCA dosages of 0 to1080 μl/l (0 to 7 E7 microbubbles/l) and ultrasound enhancementexpressed in dB relative to the baseline (0 μl/l). To measuremicrobubble stability in the ultrasound beam, 280 μl/l (1.8 E7microbubbles/l) of UCA were added to the sample container andenhancement measured once per minute over 15 minutes. Ultrasoundenhancement at each time point was then normalized to the initial signalenhancement. The ability to detect the UCA using a commercial ultrasoundscanner was also investigated using a LOGIQ® 9 ultrasound scanner with 9L probe operating in nonlinear contrast imaging mode (GE Healthcare,Milwaukee, Wis.). A bolus injection of 1 ml/l of UCA was circulatedthrough a flow phantom (model 524; ATS Laboratories, Bridgeport, Conn.)with a 6-mm-diameter vessel embedded at a depth of 2 cm in urethanerubber using a roller pump set at 250 ml/min. Ultrasound images werethen obtained every minute for 15 minutes.

Determination of Release Kinetics after Ultrasound Triggering

Oxygen release kinetics were measured using an OXYLITE® 2000 with barefiber pO₂ probe (Oxford Optronix, Oxford, United Kingdom). Ultrasoundtriggering was performed using a SonixRP scanner with a PA4-2 cardiacprobe operating at 100% acoustic output (approximately 3.6 MPa peak-peakpressure) at 4 MHz in power Doppler mode. Two milliliters ofreconstituted agent was added to 100 ml of degassed saline. Samples weretriggered with ultrasound over 20 minutes with readings obtained every30 seconds. The experimental group consisted of 2 ml of O₂ filledSE61_(O2) combined with ultrasound, while control groups consisted of 2ml of O₂ filled SE61_(O2) without ultrasound, 2 ml of nitrogen filledSE61 combined with ultrasound, and 2 ml of deionized water withultrasound. All pO₂ values were normalized to baseline levels andexpressed as change in mmHg.

Statistical Analysis

All experiments were repeated in triplicate from 3 independent samplesand averaged. Statistical significance between groups was determinedusing a one-way ANOVA with a Bonferroni multiple comparison posttest.All statistics were performed in GRAPHPAD® Prism (Version 5.0, GraphPadSoftware, San Diego, Calif.) with significance determined by α=0.05.

Results Size, Stability, and Ultrasound Enhancement

The SE61_(O2) microbubbles were successfully charged with O₂ andremained intact when reconstituted with deionized water. Reconstitutedparticles demonstrated a spherical shape and smooth morphology on lightmicroscopy as shown in FIG. 1A. These results demonstrate the presenceof microbubbles after reconstitution. Analysis of the particle sizedistribution by dynamic light scattering (FIG. 1B) showed an averagediameter of 3.1±0.1 μm with a polydispersity index of 0.89±0.18indicating a broad size distribution. Particle counting by flowcytometry (example plot shown in FIG. 1C and FIG. 1D) showed theSE61_(O2) microbubbles consisted of approximately 6.5±0.8×10⁷microbubbles/ml after suspension in 2 ml of saline.

Results of ultrasound enhancement and stability testing are shown inFIG. 2. In vitro enhancement at 5 MHz (FIG. 2 left) increased withSE61_(O2) dose, with a peak enhancement of 16.9±1.0 dB at a dose of 880μl/l (5.7 E7 microbubbles/l). As a control, freeze-dried SE61 wasreconstituted with deionized water alone (no gas), to detect thepresence of any remaining octafluoropropane. No detectable enhancementwas observed (FIG. 2 left) with enhancement levels remaining at baselinelevels (average enhancement 1.4±0.9 dB, p<0.0001 relative to SE61_(O2)).The SE61_(O2) bubbles were insonated at low level ultrasound (0.69 MPa)to determine microbubble stability (FIG. 2 right). These microbubblesdemonstrated good overall stability, with a half-life over 15 minutes.Finally, 880 μl/l of SE61_(O2) microbubbles were imaged in a flowphantom using a commercial scanner (FIG. 3). The agent demonstratedsubstantial enhancement within the 6 mm vessel lumen at a depth of 2 cmafter injection (bottom), relative to baseline (top) in both grayscaleB-mode ultrasound (left split screen), and nonlinear contrast mode(right split screen, in gold).

In Vitro Oxygen Release

Oxygen release experiments were performed to determine the ability ofSE61_(O2) to locally increase O₂ concentrations when triggered withultrasound (FIG. 4). All groups showed a gradual rise in partialpressure of O₂ (pO₂) due to gas exchange between the degassed saline andatmospheric air (average of 40 mmHg over 20 mins). However, pO₂ levelswere found to be significantly elevated over a 20 minute period whenSE61_(O2) bubbles were insonated relative to ultrasound alone,uninsonated SE61_(O2), and insonated nitrogen filled SE61 (p<0.001).This difference became apparent after 1 minute of insonation (withdifferences of 12.0, 11.4, and 7.7 mmHg respectively) and remainedconsistent throughout the full 20 minutes (23.0, 13.8, and 20.6 mmHgrespectively at 20 minutes). Untriggered SE61_(O2) showed higher pO₂levels at 20 minutes relative to triggered nitrogen filled SE61 (6.8mmHg), although their release curves were not found to be significantlydifferent (p=0.5).

In Vivo Results

Preliminary in vivo results demonstrate the ability of the UCA totemporarily elevate hypoxic tumor oxygenation levels in mice (FIGS. 6and 7). While imaging was not optimized for destruction of SE61 andtumor oxygenation appeared to be very dependent on pO₂ probepositioning, triggering of SE61_(O2) showed an increase in tumoroxygenation levels in both animals.

FIG. 6 shows example ultrasound images and tumor oxygenation levels overtime in both animals. The top ultrasound image of FIG. 6 shows thetriggering sequence of contrast arrival within the periphery of thetumor with slight peripheral enhancement denoted with two arrows. Themiddle ultrasound image shows the flash-destructive ultrasound pulseused to destroy the microbubbles, and the bottom ultrasound image showsa slight decrease in peripheral enhancement immediately aftermicrobubble destruction. The pO₂ probe introduced into the tumor throughthe catheter was observed in all images (arrow in the bottom ultrasoundimage). Both animals showed an increase in pO₂ during triggering ofSE61_(O2), although measurements appeared to be highly dependent onprobe position (FIG. 7). Ultrasound triggering in mouse 1 showed anincrease of 27.4 mmHg, with elevated tumor oxygen levels lasting 1.7 minafter injection before returning to baseline. SE61_(O2) triggering inmouse 2 resulted in a 30.4 mmHg increase, with elevated tumor oxygenlevels lasting over 4 min. Ultrasound-triggering of SE61N2 resulted inno discernible increase in oxygen partial pressure in either mouse.Untriggered SE61_(O2) (i.e., without ultrasound exposure) resulted in noincrease in oxygen partial pressure in one animal, and a brief (20 s)5.6 mmHg increase in the second animal.

Referring now to FIGS. 8A-8E, in vivo imaging in a 3 kg swine afterinjection of SE61 filled with O₂ demonstrated the enhancement of therenal vasculature. Imaging was performed using a SIEMENS® 53000ultrasound system with 9 MHz linear probe in CPS mode. Microbubblesignal is displayed in gold (left half of images), while the normalB-mode ultrasound is displayed in grayscale (right half of images).FIGS. 8A, 8B, 8C, 8D, and 8E were taken at 19, 26, 34, 44, and 54seconds after injection, respectively. The peak enhancement occurred at44-54 seconds post injection (FIGS. 8D and 8E) and lasted up to 3minutes. These data demonstrate the stability of the oxygen filledmicrobubble and ability to deliver oxygen in a large animal model.

EQUIVALENTS

Although preferred embodiments of the invention have been describedusing specific terms, such description is for illustrative purposesonly, and it is to be understood that changes and variations may be madewithout departing from the spirit or scope of the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

1. An ultrasound contrast agent (UCA) comprising an outer shell and agas core, wherein said gas core is filled with substantially pureoxygen, and wherein said outer shell comprises a first surfactant and asecond surfactant.
 2. The UCA of claim 1, wherein the gas core is filledwith about 100% pure oxygen.
 3. The UCA of claim 1, wherein the gas coreis filled with at least about 90% pure oxygen.
 4. The UCA of claim 1,wherein the first surfactant is d-α-tocopheryl polyethylene glycol 1000succinate (TPGS).
 5. The UCA of claim 1, wherein the first surfactant ispolyoxyethylene sorbitan monooleate.
 6. The UCA of claim 1, wherein thesecond surfactant is selected from the group consisting of: sorbitanesters, alkylphenol ethoxylate-based surfactants, alcoholethoxylate-based surfactants, silicone-based surfactants, alkylpoly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide), alkyl polyglucosides,fatty alcohols, cocamide MEA, cocamide DEA, and polysorbates.
 7. The UCAof claim 1, wherein the mean diameter of the UCA is in the range of 1 μmto 10 μm.
 8. The UCA of claim 1, wherein the mean diameter of the UCA isin the range of 2 μm to 7 μm.
 9. The UCA of claim 1, wherein the meandiameter of the UCA is about 3 μm.
 10. The UCA of claim 1, wherein thehalf-life of the UCA under 5 MHz ultrasound is in the range of 1 to 20minutes.
 11. The UCA of claim 1, wherein the half-life of the UCA under5 MHz ultrasound is in the range of 5 to 20 minutes.
 12. The UCA ofclaim 1, wherein the half-life of the UCA under 5 MHz ultrasound is inthe range of 10 to 20 minutes.
 13. The UCA of claim 1, wherein thehalf-life of the UCA under 5 MHz ultrasound is about 15 minutes.
 14. TheUCA of claim 1, wherein: the first surfactant is TGPS; the secondsurfactant is sorbitan monostearate; and the gas core is filled withabout 100% pure oxygen.
 15. The UCA of claim 14, wherein the meandiameter of the UCA is about 3 μm.
 16. A method of making anoxygen-filled UCA, said method comprising the steps of: a. mixing afirst surfactant and a second surfactant in phosphate buffered saline;b. heating the mixture until both surfactants are dissolved; c. coolingsaid mixture to room temperature while stirring; d. placing the mixturein a vessel in an ice bath, purging said mixture using a gas whilesonicating said mixture; e. separating microbubbles; f. adding alyoprotectant to the microbubbles; g. freeze-drying the microbubbles; h.sealing the microbubbles under vacuum; and i. refilling the microbubbleswith substantially pure oxygen.
 17. The method of claim 16, wherein: thefirst surfactant is TPGS; the second surfactant is sorbitanmonostearate; the gas is octafluoropropane; and the lyoprotectant isglucose.
 18. A method of delivering oxygen to a local area of asubject's body, comprising a. injecting a composition comprising a UCAof claim 1 into the subject's body; and b. directing ultrasoundradiation to the local area in an intensity sufficient to rupture theUCA.
 19. A method of improving effectiveness of radiotherapy andchemotherapy against cancer, comprising the steps of: a. injecting acomposition comprising a UCA of claim 1 into a subject suffering fromcancer; b. directing ultrasound radiation to a location of said cancerin an intensity sufficient to rupture the UCA; and c. applyingradiotherapy and/or chemotherapy.
 20. A method of delivering a drug to asubject, comprising the steps of: a. preparing a drug-containing UCA; b.injecting the UCA into the circulatory system of the subject; and c.directing ultrasound radiation to a location of interest with anintensity sufficient to rupture the drug-containing UCA.